Transforming growth factor-beta isoforms (TGF?1, ?2, and ?3) are secreted signaling proteins. They function as immunomodulatory factors and tumor suppressors in normal and early neoplastic cells, but in many established cancers, including androgen-independent prostate cancer, they promote tumor growth and metastasis. The therapeutic benefit of antagonizing TGF? using neutralizing antibodies and small molecule receptor kinase inhibitors has been amply demonstrated in animal models of cancer, yet no inhibitors have been approved for cancer treatment in humans. The kinase inhibitor LY2157299 has significant off-target activity and has progressed slowly through clinical trials due to safety concerns. The pan-isoform neutralizing antibody GC1008, while specific and safe, has limited efficacy. The latter may be due to i) the limited affinity of the antibody (Kd 4-10 nM) that hinder its ability to compete against the endogenous receptor complex, a heterotetramer that binds the TGF?s with affinities of 0.1-1 pM, ii) a requirement that two antibodies bind to homodimeric TGF? to completely block receptor binding, or iii) the large size of the antibody (160 kDa) and/or binding to Fc receptors that restricts its ability to penetrate the tumor and microenvironment. The objective of this study is to investigate a promising new class of TGF? inhibitors in which the ligand-binding domains of the TGF? receptors are fused together by flexible linkers. The advantages of these fusions include i) potentially higher affinities, ii) blockage of all receptor binding sites with a single inhibitor, and iii) reduced size (24-91 kDa). Through preliminary studies, four fusions have been generated. These potently inhibit TGF? activity in vitro (EC50 2 nM -1 pM) and are highly effective in suppressing primary tumor growth and distant metastases in several models, including a xenograft model of human prostate cancer. The objective of Aim 1 is to generate a series of receptor fusions of varying affinity, blockage of receptor binding sites, and size - this will allow us to investigate our hypothesis tha these parameters determine therapeutic efficacy. The PK properties of the fusions will also be evaluated in Aim 1. The primary objective of Aim 2 is to evaluate the inhibitory effect of the fusions on primary tumor growth and number and size of metastatic colonies to the major organs in two genetic mouse models with spontaneous prostate carcinogenesis in an immune-competent background and a xenograft model of human prostate cancer in an immune compromised background. To better understand how the inhibitors influence therapeutic efficacy, we will investigate their PD properties and their effects on TGF? - regulated immune suppression, angiogenesis, and EMT. The secondary objective of Aim 2 is to determine whether the fusions interfere with TGF?'s tumor suppressive and/or immune modulatory functions in immune competent mice - this will provide information as to how aggressively TGF? can be inhibited without interfering with its function in normal cells and tissues. The long-term goal is o produce fusions for neutralizing TGF?'s tumor promoting activity that are both safe and effective for use in clinical trials for advanced prostate cancer.